Biotechnology in 21st century presents the possibility of new solutions to food, environmental and health problems, with the ultimate object of promoting human prosperity. In recent years, the technology of using stem cells has been considered as a new way to treat incurable diseases. Previously, organ transplantation, gene therapy, etc., were presented for the treatment of incurable human diseases, but their efficient use has not been achieved due to immune rejection, short supply of organs, insufficient development of vectors, and an insufficient knowledge of disease genes.
For this reason, with increasing interests in stem cell studies, it has been recognized that totipotent stem cells having the ability to form all the organs by proliferation and differentiation can not only treat most of diseases but also fundamentally heal organ injuries. Also, many scientists have suggested clinical applicability of stem cells for the regeneration of all the organs and the treatment of incurable diseases, including Parkinson's disease, various cancers, diabetes and spinal damages.
Stem cells refers to cells having not only self-replicating ability but also the ability to differentiate into at least two cells, and can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells.
Totipotent stem cells are cells having totipotent properties capable of developing into one perfect individual, and these properties are possessed by cells up to the 8-cell stage after the fertilization of an oocyte and a sperm. When these cells are isolated and transplanted into the uterus, they can develop into one perfect individual.
Pluripotent stem cells, which are cells capable of developing into various cells and tissues derived from the ectodermal, mesodermal and endodermal layers, are derived from an inner cell mass located inside of blastocysts generated 4-5 days after fertilization. These cells are called “embryonic stem cells” and can differentiate into various other tissue cells but not form new living organisms.
Multipotent stem cells, which are stem cells capable of differentiating into only cells specific to tissues and organs containing these cells, are involved not only in the growth and development of various tissues and organs in the fetal, neonatal and adult periods but also in the maintenance of homeostasis of adult tissue and the function of inducing regeneration upon tissue damage. Tissue-specific multipotent cells are collectively called “adult stem cells”.
Adult stem cells are obtained by taking cells from various human organs and developing the cells into stem cells and are characterized in that they differentiate into only specific tissues. However, recently, experiments for differentiating adult stem cells into various tissues, including liver cells, succeeded and thus are receiving attention.
The multipotent stem cells were first isolated from adult bone marrow (Jiang et al., Nature, 418:41, 2002), and then also found in other various adult tissues (Verfaillie, Trends Cell Biol., 12:502, 2002). In other words, although bone marrow is the most widely known source of stem cells, the multipotent stem cells were also found in the skin, blood vessels, muscles and brains (Tomas et al., Nat. Cell Biol., 3:778, 2001; Sampaolesi et al., Science, 301:487, 2003; Jiang et al., Exp. Hematol., 30:896, 2002). However, stem cells are very rarely present in adult tissues, such as bone marrow, and such cells are difficult to culture without inducing differentiation, and thus difficult to culture in the absence of specifically screened media. Namely, it is very difficult to maintain the isolated stem cells in vitro.
Meanwhile, the results of studies on the isolation of mesenchymal stem cells from fetal tissue revealed that there are abundant mesenchymal stem cells in fetal tissue. However, the use of fetal tissue as a source of cell therapeutic agents has been limited due to ethical concerns. Mesenchymal stem cells were also isolated from umbilical cord blood (UCB) as the source of fetal mesenchymal stem cells (MSCs), but their numbers were very small, and they show poor proliferation.
On the other hand, in the case of placenta stem cells which are receiving a great deal of attention recently due to excellent differentiation potential and safety, mesenchymal stem cells can be extracted in an amount 100 times larger than that of those from umbilical cord blood. Moreover, umbilical cord blood can be used only once, and donor umbilical cord blood is additionally needed after the age of 15 years, whereas placenta stem cells can be used several times, and thus can also be used in adults. In addition, placenta stem cells can be used for a wider range of diseases. Hematopoietic stem cells of umbilical cord blood are mainly used for blood diseases, whereas placenta stem cells can be advantageously used for treating cellular injury and can be used in the future for treating many diseases, including heart failure, stroke, diabetes, osteoporosis, degenerative arthritis and the like.
However, in the case of using the placenta as the source of adult stem cells, is difficult to use placenta tissue collected immediately after delivery, whenever needed, and in fact, for the industrial application (placenta injection, placenta cosmetic products, etc.) of placenta tissue, placenta tissue collected after delivery is cold stored for use. Placenta-derived stem cells present in an undifferentiated state can be obtained in a large amount only from the placenta collected immediately after delivery, and it is very difficult to obtain a large amount of adult stem cells from placenta tissue several hours after delivery or particularly from placenta tissue that has been cold-stored for a long period of time.
For this reason, in order to use placenta-derived adult stem cells in industrial applications there is an urgent need to develop technologies for preparing a large amount of adult stem cells from cold-stored placenta tissue or the like. Among adult stem cells, it is known that adult stem cells derived from amniotic epithelial cells possess properties most similar to the properties of embryonic stem cells, and thus have the capability to differentiate into various cells, while they are clinically safe, because they do not cause cancer upon in vivo transplantation, unlike embryonic stem cells (Miki et al., Stem Cell, 23:1549, 2005). The adult stem cells derived from amniotic epithelial cells are more likely to proceed to apoptosis during the culture and subculture of single cells, unlike adult stem cells derived from other placenta tissues. For this reason, it is difficult to induce proliferation of undifferentiated adult stem cells derived from amniotic epithelial cells in large numbers using conventional culture methods (http://www.cellapplications.com/HumanCells/HPlEpC.htm).
Accordingly, the present inventors have made many efforts to prepare a large amount of undifferentiated adult stem cells derived from amniotic epithelial cells for practical applications thereof and, as a result, have found that a large amount of stem cells derived from amniotic epithelial cells can be obtained from cold-stored placenta tissue by using DTT and a ROCK inhibitor to isolate and culture amniotic epithelial cells and changing the composition of culture medium, thereby completing the present invention.